NTB-A, a surface molecule involved in natural killer cells activity

ABSTRACT

The present invention relates to a novel protein, termed NTB-A, nucleic acid molecules encoding the same and uses thereof. The invention also relates to methods of regulating Natural Killer cells activity by regulating the activity of NTB-A in vitro, ex vivo or in vivo. The invention also comprises methods of screening active compounds using NTB-A or fragments thereof, or nucleic acid encoding the same, or recombinant host cells expressing said polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/484,139, filed Jan. 16, 2004, which is the national stage ofinternational application No. PCT/EP02/07945, filed Jul. 17, 2002.

The present invention relates to a novel protein, termed NTB-A, nucleicacid molecules encoding the same and uses thereof. The invention alsorelates to methods of regulating an immune response in a subject byregulating the activity of NTB-A in vitro, ex vivo or in vivo. Theinvention also relates to methods of regulating the activity of immunecells, particularly of Natural Killer cells, T lymphocytes and/or Blymphocytes by regulating the activity of NTB-A in vitro, ex vivo or invivo The invention also comprises methods of screening active compoundsusing NTB-A or fragments thereof, or nucleic acid encoding the same, orrecombinant host cells expressing said polypeptide.

Important advances have recently been made in our understanding of therole of human NK cells in host defenses. Progress is mainly consequentto the discovery of a series of receptors, expressed at the NK cellsurface, regulating NK cell functions. Some of these receptors inhibitNK cells by monitoring the expression of MHC class I molecules on normalcells (1-3). Other receptors are responsible for NK cell activation (4).This occurs when NK cells interact with cells that, as a consequence ofviral infection or tumor transformation, do not express, or expressinadequate amounts of, MHC class I molecules (5). In humans, differenttriggering receptors have been identified. NKp46 (6,7), NKp30 (8), andNKp44 (9, 10), collectively termed “natural cytotoxicity receptors”(NCRs) (11) are selectively expressed by NK cells and cooperate witheach other in the induction of natural cytotoxicity. NCRs arecharacterized by a coordinated surface expression and a directcorrelation exists between their surface density and the ability of NKcells to kill various tumors (12). NCRs are all members of the Igsuperfamily (IG-SF), display low degree of similarity to each other, andare coupled to different signal transducing adaptor proteins includingCD3ζ, FcεRIγ, and KARAP/DAP12 (8-10).

Another triggering receptor expressed by NK cells is represented byNKG2D, a C-type lectin surface molecule encoded within the “NK genecomplex” on human chromosome 12 (13). Upon recognition of thestress-inducible MICA/B molecules (14) on target cells, NKG2D stronglyenhances the NK-mediated cytotoxicity (14, 15). Different from NCR,NKG2D is also expressed by virtually all TCR-γ/δ+ as well as by CD8TCR-α/β+ cells. Its function requires the association with a newlyidentified signalling subunit termed DAP 10 (16) or KAP10 (17). Asrecently shown, NKG2D can complement the role of NCRs in tumor celllysis, their relative involvement being primarily dependent on the typeof target and/or effector cells analyzed (15).

Notably, optimal NK cell triggering may require the function of anothermolecule termed 2B4 (CD244; references 18-20). This molecule isexpressed not only by NK cells, but also by monocytes and a subset ofCD8 T cells. In NK cells, the mAb-mediated cross-linking of 2B4 or itsengagement by CD48 (i.e., the 2B4 ligand) results in the enhancement ofcytolytic activity (18-22). Analysis at the clonal level has clearlyshown that this effect is restricted to NK cells expressing high NCRsurface density (NCR bright). Moreover, mAb-mediated modulation of NCRsresults in NK cell unresponsiveness to 2B4 engagement, although itssurface expression is not affected. Thus, the ability of 2B4 to induceNK cell activation appears to be dependent on the co-engagement of maintriggering receptors including NKp46. This supports the notion that 2B4may function as a co-receptor rather than as a true receptor (23). 2B4is a member of the CD2 subfamily of the Ig-SF which also includes CD48,CD58, CD84, CD150 (also termed SLAM), and CD299 (Ly9) (24). 2B4 ischaracterized, in its extracellular portion, by a membrane-distal Ig Vdomain and a membrane-proximal IgC2 domain. The transmembrane regionlacks charged amino acids involved in the association with immunetyrosine-based activating motif (ITAM)-bearing signal transducingpolypeptides (21, 22, 25-27). Interestingly, the cytoplasmic tailcontains four tyrosine-based motifs (TxYxxI/V) that undergophosphorylation upon sodium pervanadate treatment (27, 28) or celltriggering with anti-2B4 mAb (29). Different polypeptides have beenshown to associate with 2B4 and to participate in the 2B4-mediatedsignal transduction pathway. Thus, upon tyrosine phosphorylation, 2B4associates with a small cytoplasmic polypeptide termed Src homology 2domain-containing protein (SH2D1A; references 27 and 28) also referredto as SLAM-associated protein (SAP [30]). Controversy still exists onwhich of the SH2-containing phosphatases (SHP) binds to 2B4. Although2B4/SHP-2 association has been described in cell transfectants (27), innormal NK cells, only 2B4/SHP-1 association could be detected (28).Recently, it has been shown that, in normal NK cells, 2B4 constitutivelyassociates with the linker for activation of T cells (LAT [29]).mAb-mediated cross-linking of 2B4 results in tyrosine phosphorylation ofLAT and recruitment of PLCγ and Grb2 intracytoplasmic signallingmolecules (29).

A dramatically altered function of 2B4 has recently been detected inindividuals affected by the X-linked lymphoproliferative disease (XLP[28, 31, 32]). XLP is a severe inherited immune deficiency,characterized by the inability to control EBV infection resulting infulminant infectious mononucleosis or lymphoma (33, 34). The geneticbasis of XLP, i.e., critical mutations in the SH2D1A encoding gene (30,35, 36) has been identified. Due to these mutations, in XLP-NK cells 2B4fails to associate with SH2D1A but associates with SHP-1 and mediatesinhibitory (rather than activating) signals (28). Thus, the engagementof 2B4 with CD48 expressed at high densities on EBV-infected cellsresults in a sharp inhibition of the NK-mediated cytotoxicity.

The present invention stems from the identification, cloning andfunctional characterization of a novel NK cells surface molecule, termedNTB-A.

NTB-A, a 60-kD glycoprotein, is expressed by human NK, T, and Blymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-Aresults in the induction of NK-mediated cytotoxicity. Similar to 2B4(CD244) functioning as a co-receptor in the NK cell activation, NTB-Aalso triggers cytolytic activity only in NK cells expressing highsurface densities of natural cytotoxicity receptors. This indicates thatNTB-A may function as a co-receptor in the process of NK cellactivation. Molecular cloning of the cDNA coding for NTB-A moleculerevealed a novel member of the immunoglobulin superfamily belonging tothe CD2 subfamily. NTB-A is characterized, in its extracellular portion,by a distal V-type and a proximal C2-type domain and by a cytoplasmicportion containing three tyrosine-based motifs. NTB-A undergoes tyrosinephosphorylation and associates with the Src homology 2 domain-containingprotein (SH2D1A) as well as with SH2 domain-containing phosphatases(SHPs). Importantly, analysis of NK cells derived from patients withX-linked lymphoproliferative disease (XLP) showed that the lack ofSH2D1A protein profoundly affects the function not only of 2B4 but alsoof NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather thanactivating signals. These inhibitory signals are induced by theinteraction of NTB-A with still undefined ligands expressed onEpstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediatedmasking of NTB-A can partially revert this inhibitory effect while amaximal recovery of target cell lysis can be obtained when both 2B4 andNTB-A are simultaneously masked. Thus, the altered function of NTB-Aappears to play an important role in the inability of XLP-NK cells tokill EBV-infected target cells.

An object of this invention resides in a nucleic acid molecule encodinga NTB-A polypeptide.

A further aspect of this invention resides in an isolated NTB-Apolypeptide.

A further aspect of this invention is an antibody that binds a NTB-Apolypeptide.

Still a further aspect of this invention is a method of regulating animmune response in a subject comprising administering to the subject acompound that regulates the activity of NTB-A.

The invention also relates to methods of screening compounds comprisingdetermining the ability of a test compound to regulate the activity ofNTB-A.

The invention also relates to a pharmaceutical composition comprising acompound that regulates the activity of NTB-A and a pharmaceuticallyacceptable vehicle or carrier.

The invention also relates to methods of diagnosing a dysfunction in asubject, comprising determining the presence of a mutation or alterationin a NTB-A gene or RNA, or determining the presence or amount of a NTB-Apolypeptide.

The invention is particularly suited to treat or diagnose pathologies inwhich the activity of immune cells is involved, particularly NK cells, Tcells and/or B cells. The invention is particularly suited to treat ordiagnose pathologies in which the activity of NK cells is involved suchas viral infections, tumor or other proliferative diseases, autoimmunediseases, etc. The invention can be used, by way of example, to treatpatients with X-linked lymphoproliferative disease.

Nucleic Acid Molecules

A particular aspect of this invention is a nucleic acid moleculeselected from:

-   -   a) a nucleic acid encoding a polypeptide comprising amino acid        sequence SEQ ID NO:2;    -   b) a nucleic acid which hybridises to the nucleic acid a) or to        a portion thereof, said portion comprising at least 30        contiguous nucleotides of a nucleic acid a),    -   c) a complementary strand of a nucleic acid molecule of a) or        b), and    -   d) a fragment of a nucleic acid molecule of a), b) or c), said        fragment comprising at least 9 contiguous nucleotides.

In a particular embodiment, the invention relates to a nucleic acidmolecule comprising a sequence encoding a polypeptide comprising aminoacid sequence SEQ ID NO:2 or a polypeptide comprising at least 5contiguous amino acid residues of SEQ ID NO:2.

The invention particularly encompasses any nucleic acid moleculeencoding a human NTB-A protein or polypeptide, as defined above.

The nucleic acid molecule can be a DNA or a RNA molecule. It can be agDNA, cDNA or a synthetic or semi-synthetic DNA, e.g., comprising addedsequences such as introns, biased codons, etc. The nucleic acid may beprepared by any conventional technique, such as by artificial synthesis,cloning, or combinations thereof.

A preferred, specific embodiment is a nucleic acid comprising SEQ IDNO:1 or a portion thereof comprising at least 9 contiguous nucleotidesthereof, or a complementary strand thereof.

As indicated, the invention encompasses nucleic acid molecules thathybridises to a nucleic acid encoding a polypeptide comprising aminoacid sequence SEQ ID NO:2, or to a portion thereof, said portioncomprising at least 30 contiguous nucleotides of a nucleic acid.Hybridization can be performed either under stringent or non-stringentconditions, preferably under stringent conditions. Typically,encompassed nucleic acids are those hybridising to the above sequencesunder the following conditions: incubation at 42° C. in 50% formamide,5×SSPE, 5× Denhardt's solution, 0.1% SDS (1×SSPE is composed of 0.15MNaCl, 10 mM NaH₂PO₄, 1.3 mM EDTA, pH 7.4). Stringent conditions are alsodescribed in ref. 37. The nucleic acid should hybridize to the entirecoding sequence or, as indicated, to at least a portion thereofcomprising 30 contiguous nucleotides. It is believed that such a lengthensures high specificity of hybridization. The hybridizing nucleic acidpreferably encodes a polypeptide having a receptor function, or a partthereof.

Preferred fragments d) of the above nucleic acid molecules comprise atleast 15 contiguous nucleotides, even more preferably at least 21nucleotides.

The above nucleic acid molecules may be used to produce a NTB-Apolypeptide in vitro, ex vivo or in vivo, to amplify the nucleic acid inany appropriate cell, as a probe or as a primer, for instance. Thesenucleic acids may also be used as antisense molecules (or to designantisense molecules) to regulate NTB-A expression in a cell, in vitro,ex vivo or in vivo.

In this respect, an object of this invention resides in a nucleic acidprobe, wherein said probe is complementary and specifically hybridizesto a nucleic acid molecule as defined above. The probe is preferably asingle-strand molecule, comprising, preferably, from 9 to about 300nucleotides. The probe may comprise the full nucleic acid sequenceencoding a NTB-A polypeptide. The probe can be specific for variants,mutants or deletants and specifically detect such species in a sample.The probe may be labelled, by any conventional means such asradioactive, enzymatic, fluorescent, luminescent, chemical, etc. Theprobe can be used to detect the presence of a NTB-A gene or RNA in asample, particularly the presence of a mutation or alteration in a NTB-Agene or RNA in a sample.

The invention also encompasses a pair of nucleic acid primers, whereinat least one primer of said pair is complementary and specificallyhybridizes to a nucleic acid molecule as defined above. Such a pair ofprimer can be used to amplify, in a sample, all or part of a gene or RNAcoding for NTB-A. Amplification can be performed by PCR, as described inthe examples, or by any other known technique. The primer is preferablya single-stranded nucleic acid molecule of less than about 50nucleotides in length, typically of between 10-50 nucleotides in length.

An antisense molecule is any nucleic acid that specifically hybridisesto a NTB-A gene or RNA and prevents or reduces transcription ortranslation thereof. The antisense may be DNA or RNA, single- ordouble-stranded, and preferably comprises a portion of a nucleic acidsequence as described above, typically at least 30 nucleotides thereof.

Vectors

The invention relates to any vector comprising a nucleic acid moleculeas defined above. The vector may be any plasmid, phage, episome,artificial chromosome, virus, etc. Typical examples include plasmidvectors, such as those derived from commercially available plasmids(pUC, pBR322, pcDNA, etc.). Other preferred vectors are viruses,particularly recombinant (replication-defective) viruses, such as thosederived from retroviruses, baculoviruses, lentiviruses, AAVs,adenoviruses, herpesviruses, etc. The vectors can be prepared byconventional techniques, e.g., by ligating the NTB-A nucleic acidmolecule into appropriate cloning site of the vector. The vector mayfurther comprise regulatory sequences, such as promoters, terminators,enhancers, silencers, etc., origin(s) of replication, marker genes, etc.Typical examples of promoters include promoters allowing constitutive orregulated or tissue-selective expression, weak or strong promoters, ofcellular, viral or synthetic origin, such as RVS-LTR, SV40-IE, CMV-IEpromoter, promoters of domestic genes (PGK, etc.), promoters of bacteriaor phage (T7, Lac, Trp, etc.), etc. The vectors can be used to express anucleic acid of this invention in vitro, ex vivo or in vivo. Inparticular, the vectors can be used to produce a NTB-A polypeptide in acell, in vitro or ex vivo, as well as directly in vivo, in a genetherapy program.

Polypeptides

An other object of this invention is a NTB-A polypeptide. Within thecontext of the present invention, a NTB-A polypeptide designates anypolypeptide comprising SEQ ID NO:2, as well as any variants, derivativesor homologs thereof, including any naturally-occurring derivativesthereof or homologs isolated from various mammalian species (includingrodents, equine, bovine, etc.). Variants more generally encompass anypolypeptide having one or several amino acid modifications as comparedto SEQ ID NO:2, including mutation(s), deletion(s), insertion(s) and/orsubstitution(s), alone or in various combinations(s). More preferably, aNTB-A polypeptide is a polypeptide having at least 70% identity, morepreferably at least 80%, further preferably at least 90% identity withthe amino acid sequence of SEQ ID NO: 2 or to a fragment thereof.Identity can be determined using known methods and commercial computerprograms, such as CLUSTAL or BLAST (NCBI). Various search or alignmentparameters may also be used. In a preferred embodiment, percentage aminoacid (or nucleic acid) identity is determined using the CLUSTAL-Wprogram (Compugen).

In a specific embodiment, the invention relates to a polypeptidecomprising amino acid sequence SEQ ID NO:2 or a fragment thereofcomprising at least 5 contiguous amino acid residues of SEQ ID NO:2.

In an other specific embodiment, this invention relates to a polypeptidecomprising amino acid sequence SEQ ID NO:2 having an extra alanineresidue at position 266, or a fragment thereof comprising at least 5contiguous amino acid residues. The inventors have indeed identified anallelic isoform of NTB-A comprising an extra codon (CAG) encoding analanine residue at position 266 of SEQ ID NO:2. The resulting NTB-Apolypeptide thus comprises 332 amino acids.

Polypeptide fragments may comprise epitopes, particularly B or T cellsepitopes, functional domains or parts of human NTB-A, including portionsof the polypeptide involved in an interaction with other proteins ormolecules within the cell, phosphorylation domains, etc. A polypeptidefragment of this invention generally comprises less than 300 aminoacids, even preferably less than 250 amino acids.

A particular polypeptide of this invention is a polypeptide comprisingamino acid residues 22 to 331 of SEQ ID NO:2 or a variant thereof havingan extra alanine residue at position 266. Residues 22-331 correspond toa mature NTB-A polypeptide.

An other polypeptide of this invention is a polypeptide comprising thesignal peptide region of NTB-A, particularly amino acid residues 1 to 21of SEQ ID NO:2.

An other polypeptide of this invention is a polypeptide comprising theextra-cellular region of NTB-A, particularly amino acid residues 22 to226 of SEQ ID NO:2, or a fragment thereof comprising at least 5contiguous amino acids. More preferred fragments comprise typicallybetween 5 and 50 consecutive amino acids, even more preferably between 6and 40. This region is responsible for the interaction of NTB-A withputative ligands on target cells and is particularly useful to regulatethe activity of NTB-A or to screen for ligands (including the endogenousligands) or modulators of NTB-A activity. Sub-fragments thereof can beused as well.

An other polypeptide of this invention is a polypeptide comprising thetransmembrane region of NTB-A, particularly amino acid residues 227 to248 of SEQ ID NO:2, or a fragment thereof comprising at least 5contiguous amino acids. More preferred fragments comprise typicallybetween 5 and 20 consecutive amino acids, even more preferably between 6and 15.

An other polypeptide of this invention is a polypeptide comprising theintra-cytoplasmic region of NTB-A, particularly amino acid residues 249to 331 of SEQ ID NO:2 or a variant thereof having an extra alanine atposition 266, or a fragment thereof comprising at least 5 contiguousamino acids. More preferred fragments comprise typically between 5 and50 consecutive amino acids, even more preferably between 6 and 40. Thisregion is responsible for the interaction of NTB-A with putativesignaling molecules or receptors with NK, B or T cells and is useful toregulate the activity of NTB-A or to screen for binding partners ormodulators of NTB-A activity. Sub-fragments thereof can be used as well.In this regard, a further polypeptide of this invention is a polypeptidecomprising functional portions of NTB-A, such as the SH2D1A bindingdomain or tyrosine residues, particularly amino acid residues 271-276(LEYVSV), 282-287 (TVYASV) or 306-311 (TIYSTI) of SEQ ID NO:2, or largerpolypeptide fragments of NTB-A comprising said residues.

The invention generally relates to any polypeptides comprising an aminoacid sequence encoded by a nucleic acid molecule as defined above.

The invention also includes a soluble NTB-A polypeptide.

The polypeptides of this invention may be attached to any heterologoussequence or moiety, such as stabilizing agent, a marker, a tag, atargeting moiety, a drug, a cytokine, a toxin, etc. They may also beattached or immobilized to solid supports, such as columns, beads, etc.The polypeptides of this invention are preferably in isolated orpurified form (i.e., not in their natural environment) or expressed fromrecombinant host cells. They may be combined to other active moleculesor adjuvants or solvents. The polypeptides can be used to produceantibodies, modulate an immune response, regulate the activity of NTB-Ain vitro, ex vivo or in vivo, screen for compounds that modulate NTB-Aactivity, etc.

Host Cells

The present invention also relates to host cells comprising a nucleicacid molecule or a vector as defined above. In a preferred embodiment,the host cell comprises a nucleic acid molecule or a vector as definedabove and expresses a polypeptide as defined above. The polypeptide ispreferably expressed at the surface of said cell. The polypeptide mayalso be expressed within the cell cytoplasm or released from the cellsby any means. The polypeptide may, in particular, be expressed as asoluble or secreted molecule, by removing portions thereof (e.g., the TMand/or cytoplasmic domains).

The recombinant cell can be any cultivable cell, such as prokaryotic oreukaryotic cells, including bacteria, yeasts, insect cells, plant cells,mammalian cells, etc. The cell may be a cell line or a primary culture.Typical examples include E. coli, Saccharomyces, Kluyveromyces, insectcells, mammalian fibroblasts or embryonic cells, HEK, CHO, COS, 3T3,293, etc. It should be understood that the invention shall not belimited to any specific type of host cell.

In a preferred embodiment, the host cell is a cell that does notnaturally express a human NTB-A polypeptide. Such host cell can be usedadvantageously in screening assays, with increased selectivity.

An other object of this invention is a method of preparing cellsexpressing a NTB-A polypeptide, said method comprising introducing intocells in vitro a nucleic acid molecule or a vector as defined above andselecting the cells or their progeny which express the polypeptide.Introduction of the nucleic acid or vector may be accomplished byvarious known means, such as by direct DNA transfer, lipid-mediatedtransfection, calcium-phosphate precipitation, electroporation, etc. Thecells may be cultured in any suitable media and stored under anyconventional methods.

Antibodies

The present invention also relates to an antibody that binds to apolypeptide as defined above. The antibody is preferably an antibodyobtained by immunizing an animal with a polypeptide as defined above orwith a host cell as defined above. In this regard, an object of thisinvention also includes a method of preparing an antibody, said methodcomprising injecting to a non-human mammal a polypeptide as definedabove and collecting the antibody, serum or antibody-producing cells insaid mammal.

The antibody may be a polyclonal or a monoclonal antibody as well asfragments and derivatives thereof, in particular fragments andderivatives of said antibodies having substantially the same antigenicspecificity. These include antibody fragments (e.g., Fab, Fab′2, CDRs,etc), humanized antibodies, polyfunctional antibodies, Single Chainantibodies (ScFv), etc. These may be produced according to conventionalmethods, including immunization of an animal and collection of serum(polyclonal) or spleen cells (to produce hybridomas by fusion withappropriate cell lines).

Methods of producing monoclonal antibodies from various speciesincluding mice, rodents, primates, horses, pigs, rabbits, poultry, etc.may be found, for instance, in Harlow et al (Antibodies: A laboratoryManual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495).Briefly, these methods comprise immunizing an animal with the antigen,followed by a recovery of spleen cells which are then fused withimmortalized cells, such as myeloma cells. The resulting hybridomasproduce the monoclonal antibodies and can be selected by limit dilutionsto isolate individual clones.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in Ward et al (Nature 341(1989) 544).

Methods of producing polyclonal antibodies from various species, aslisted above, may be found, for instance, in Vaitukaitis et al., 1971.Briefly, the antigen is combined with an adjuvant (e.g., Freund'sadjuvant) and administered to an animal, typically by subcutaneousinjection. Blood samples are collected and immunoglobulins or serum areseparated.

Fab or F(ab′)2 fragments may be produced by protease digestion,according to conventional techniques. Humanized antibodies can beprepared as described previously (Jones 1986).

Preferred antibodies of this invention are prepared by immunization witha NTB-A polypeptide as described above, preferably with a polypeptide ofSEQ ID NO:2 or a variant having an extra alanine residue at position266, or a fragment thereof, particularly a fragment comprising all orpart of the extracellular domain of NTB-A. In this regard, particularlypreferred antibodies of this invention are (monoclonal) antibodies thatspecifically bind an epitope comprised in amino acid residues 22-226 ofSEQ ID NO: 2.

Most preferred antibodies of this invention are functional antibodies,i.e., antibodies that regulate the activity of NTB-A, more particularlyantibodies that can activate or block the interaction of NTB-A with atarget cell. Such antibodies can be further selected by contacting anantibody as disclosed above with a NK cell and a target cell, and bydetermining the ability of said antibody to block or to activateNK-mediated target cell lysis. The present invention shows that suchfunctional (e.g., blocking) antibodies can be produced and used toregulate the activity of immune cells.

The antibodies may be coupled to heterologous moieties, such as toxins,labels, drugs or other therapeutic agents, covalently or not, eitherdirectly or through the use of coupling agents or linkers. Labelsinclude radiolabels, enzymes, fluorescent labels, magnetic particles andthe like. Toxins include diphtheria toxins, botulinum toxin, etc. Drugsor therapeutic agents include lymphokines, antibiotics, antisense,growth factors, etc. Methods of using such heterologous moieties areillustrated, for instance, in U.S. Pat. Nos. 4,277,149 and 3,996,345.

The antibodies of this invention have various applications, includingtherapeutic and diagnostic uses, and they may be used as well forimmuno-purification or detection of a NTB-A polypeptide in a sample(e.g., plasma or serum samples), for instance. In particular, they canbe used in vitro in screening assays as will be described below, or todetect or quantify the presence (or amounts) of NTB-A polypeptide in asample collected from a subject, typically a blood sample from amammalian, specifically a human subject.

Screening

The present invention provides a novel molecule involved in the activityof NK cells and potentially of T and B lymphocytes, and in theregulation of the immune system. This molecule, as well as thecorresponding nucleic acids, host cells, plasmids, and binding partnersthereof, can be used to screen for compounds, particularly biologicallyactive compounds, especially in the area of immune regulation and NKregulation.

In particular, the invention also relates to methods of screeningcompounds comprising determining the ability of a test compound toregulate the activity of NTB-A. Within the context of the presentinvention, the term “activity of NTB-A” includes the expression of NTB-Aas well as the biological activity of a NTB-A polypeptide. Regulatingthe activity of NTB-A thus includes the regulation of NTB-A synthesis(e.g., transcription, translation, trafficking, export to the cellmembrane, post-translational modification, etc.) as well as theregulation of the biological activity of NTB-A (e.g., its interactionwith a ligand, the cross linking or internalisation of NTB-A, thephosphorylation or translocation of NTB-A, the association of NTB-A withan other cellular partner, etc.).

Preferred compounds are those that selectively regulate the activity ofNTB-A, i.e., that essentially do not directly interfere with an other NKcell receptor, although secondary effects may be induced.

In a particular embodiment, the method comprises determining the abilityof a test compound to:

-   -   bind to NTB-A,    -   activate NTB-A    -   inhibit NTB-A    -   inhibit NTB-A synthesis    -   stimulate NTB-A synthesis    -   modulate NTB-A binding to its putative endogenous ligand,    -   modulate (e.g., block or activate) the interaction of NTB-A (or        cells expressing NTB-A such as NK cells, T lymphocytes, B        lymphocytes, recombinant cells, etc) with target cells,    -   modulate (e.g., block or activate) the NTB-A-mediated NK cell        induced target cell lysis,    -   modulate the interaction of NTB-A with SH2D1A or    -   modulate the interaction of NTB-A with SHP

In a particular embodiment, this invention lies in a method ofselecting, screening or characterizing a compound, said methodcomprising contacting a test compound with a polypeptide as definedabove and determining the ability of said test compound to bind to saidpolypeptide. The polypeptide may be isolated or expressed by a cell,particularly at the surface of said cell.

The invention thus also relates to a method of selecting, screening orcharacterizing a compound, said method comprising contacting a testcompound with a host cell as defined above (said host cell expressing aNTB-A polypeptide) and determining the ability of said test compound tobind to said polypeptide.

The ability of a test compound to bind to a NTB-A polypeptide may bedetermined by various techniques known in the art. In vitro, the bindingmay be determined by electrophoresis, SPA, FRET, etc. or by competitionwith a labelled ligand of a NTB-A polypeptide. In this regard, in apreferred embodiment binding is determined by measuring the ability ofthe test compound to modulate the binding of a labelled ligand. Thelabelled ligand may be, for instance, an antibody.

The invention also relates to a method of selecting, screening orcharacterizing a compound, said method comprising contacting a testcompound with a host cell as defined above (said host cell expressing aNTB-A polypeptide) and determining the ability of said test compound toinduce a biological response characteristic of a NTB-A polypeptide. Thebiological response may be, for instance, an association with bindingpartners, a cytolytic activity, etc.

In a particular embodiment, the invention comprises contacting a testcompound with a NK cell in the presence of an antibody specific forNTB-A and determining the activity said test compound by measuring thecytolytic activity of said NK cells.

In a further embodiment, the invention relates to a method of selecting,screening or characterizing a compound comprising contacting a testcompound with a NTB-A polypeptide in the presence of a binding partnerthereof and assessing the capacity of said test compound to modulate theinteraction between said NTB-A polypeptide and said binding partner. Thebinding partner is, for instance, all or part of SH2D1A or of SHP. Theinvention indeed shows that NTB-A comprises, within theintra-cytoplasmic domain, regions allowing binding to signallingmolecules such as SH2D1A or of SHP. The invention further shows thatsuch an interaction indeed occurs in physiological conditions. Thescreening of compounds having the ability to interfere with saidinteraction thus provides molecules having the ability to regulate theactivity of NTB-A. Such a screening can be performed in vitro, bycontacting a NTB-A polypeptide (or a portion thereof comprising all orpart of the intra-cytoplasmic domain thereof) with the test compound andthe binding partner.

In an other embodiment, the binding partner is a ligand of NTB-A,particularly a ligand expressed by target cells. The screening ofcompounds having the ability to interfere with said interaction thusprovides molecules having the ability to block or stimulate NKcell-mediated target cell lysis. Such a screening can be performed invitro, by contacting a NTB-A polypeptide (or a portion thereofcomprising all or part of the extra-cytoplasmic domain thereof) with thetest compound and the ligand or a target cell expressing the ligand.

In a particular embodiment, a screening method comprises (i) determiningthe ability of a test compound to bind to a NTB-A polypeptide and (ii)determining the ability of a test compound selected in (i) to regulate(e.g., block, reduce or stimulate) NK cell-mediated target cell lysis.

The test compound may be any synthetic compound, including organicproducts, lipids, peptides, proteins, nucleic acids (e.g., antisense),etc. The assay may be performed in any suitable device, such as plates,tubes, dishes, flasks, etc. Typically, the assay is performed inmulti-wells plates. Several test compounds can be assayed in parallel.Furthermore, the test compound may be of various origin, nature andcomposition. It may be any organic or inorganic substance, isolated orin mixture with other substances. The compounds may be all or part of acombinatorial library of products, for instance.

Uses

The invention discloses a novel protein and corresponding screeningmethods. The protein is involved in the regulation of immune functionand its modulation or regulation can thus produce a regulation of animmune function in vivo. In particular, compounds that regulate theactivity of NTB-A may be used to regulate the activity of NK cells, Bcells or T cells, particularly to stimulate or inhibit the activity ofNK cells, more specifically the NK cells-mediated cytolysis. Suchcompounds are thus suitable for use in the treatment, prevention ordiagnosis of pathologies in which the activity of NK cells is involved,such as viral infections, tumor or other proliferative diseases,autoimmune diseases, etc. The invention can be used, by way of example,to treat patients with X-linked lymphoproliferative disease.

An object of this invention thus lies in a method of regulating animmune response in a subject comprising administering to the subject acompound that (selectively) regulates the activity of NTB-A. Theinvention also relates to the use of a compound that (selectively)regulates the activity of NTB-A in the manufacture of a medicament forregulating an immune response in a subject.

The invention also relates to a pharmaceutical composition comprising acompound that regulates the activity of NTB-A and a pharmaceuticallyacceptable vehicle or carrier. Particular compositions comprises acompound that stimulates the activity of NTB-A or that inhibits theactivity of NTB-A.

The compositions of this invention may further comprise a compound thatregulates an other NK cell receptor, particularly the 2B4 receptor. Suchcompositions indeed provide significant effect in vivo, particularly inXLP disease, by restoring NK or T cell activity in such patients. Aparticular object of this invention thus resides in a composition thatcomprises a compound that regulates a NTB-A receptor and a compound thatregulates a 2B4 receptor, for combined, sequential or separated use. Thecompound is preferably a compound that inhibits or reduces NTB-Aactivity, particularly an antibody or a molecule selected or identifiedby a method as described above. The examples provided below illustratethe efficacy of such a combined treatment to restore NK cell activity ina patient with XLP disease.

The compounds may be administered according to various routes, such asby intravenous, intra-arterial, intra-muscular, intra-dermic,intra-peritoneal, etc. The compounds may be used in various dosages thatcan be adapted by the skilled person.

The compounds are particularly suited to stimulate an immune response ina human subject, particularly to stimulate NK cells in a human subject.

The invention also relates to methods of diagnosing a dysfunction in asubject, comprising determining the presence of a mutation or alterationin a NTB-A gene or RNA, or determining the presence or amount of a NTB-Apolypeptide. These methods may be performed using antibodies or nucleicacid primers or probes as described above. The method can be performedwith any sample derived from a subject, such as serum, blood, plasma,other biological fluids, tissue samples, etc.

Further aspects and advantages of the present invention will bedisclosed in the following examples, which should be regarded asillustrative and not limiting the scope of this applications. All citedreferences are incorporated therein by reference.

LEGEND TO THE FIGURES

FIG. 1. Biochemical characterization of NTB-A molecules. NK cell, TCRgamma delta cell and thymocyte (Thy) populations that had been surfacelabeled analysis of NTB-A molecules in redirected killing assay and inNK-mediated killing of EBV target cells. (a) Representative NKp46 bright(MX367, CC16) and NKp46 dull (HER12, HER3) NK cell clones were analyzedin a redirected killing assay against (FcR) P815 murine target cellseither in the absence or in the presence of mAbs specific for theindicated molecules. All the mAbs were of the IgG1 subclass. (b) Therepresentative NKp46 bright clone (MOA 110) either untreated ormodulated with anti-NKp46 (KL247, IgM) mAb was tested for cytolyticactivity in a redirected killing assay against murine P815 target cellseither in the absence or in the presence of mAbs specific for theindicated molecules. In both experiments the E/T ratio used was 8:1. (c)Two representative NKp46 bright NK cell clones (MX367 and MOA110) wereanalyzed for cytolytic activity against the LCL 721.221 EBV cell line(HLA class I CD48 FcR) either in the absence or in the presence of mAbsspecific for the indicated molecules. The E/T ratio used was 2:1. (d)The NK92 NK cell line was analyzed for cytolytic activity against the(FcR) P815 murine target cells or the LCL 721.221 EBV cell line, eitherin the absence or in the presence of mAbs specific for the indicatedmolecules. The E/T ratio used was 5:1. The results are representative ofseven independent experiments. The standard deviation of the mean of thetriplicates was 4%.

FIG. 2. Functional analysis of NTB-A molecules in redirected killingassay and in NK-mediated killing of EBV+ target cells. (a)Representative NKp46 bright (MX367, CC16) and NKp46 dull (HER12, HER3)NK cell clones were analyzed in a redirected killing assay against(FcγR+) P815 murine target cells either in the absence or in thepresence of mAbs specific for the indicated molecules. All the mAbs wereof the IgG1 subclass. (b) The representative NKp46 bright clone (MOA110)either untreated or modulated with anti-NKp46 (KL247, IgM) mAb wastested for cytolytic activity in a redirected killing assay againstmurine P815 target cells either in the absence or in the presence ofmAbs specific for the indicated molecules. In both experiments the E/Tratio used was 8:1. (c) Two representative NKp46 bright NK cell clones(MX367 and MOA110) were analyzed for cytolytic activity against the LCL721.221 EBV cell line (HLA class I-CD48+, FcγR⁻) either in the absenceor in the presence of mAbs specific for the indicated molecules. The E/Tratio used was 2:1. (d) The NK92 NK cell line was analyzed for cytolyticactivity against the (FcgR+) P815 murine target cells or the LCL 721.221EBV cell line, either in the absence or in the presence of mAbs specificfor the indicated molecules. The E/T ratio used was 5:1. The results arerepresentative of seven independent experiments. The standard deviationof the mean of the triplicates was <4%.

FIG. 3. Analysis of the NTB-A-specific signal transduction pathway. (a)Cell lysates derived from a polyclonal NK cell population eitheruntreated (−) or treated (+) with sodium pervanadate (100 M, 10 min,37C), were sequentially immunoprecipitated (IP) with anti-2B4 andanti-NTBA mAbs. Samples were analyzed under reducing conditions in 8%SDS-PAGE and probed with anti-phosphotyrosine (anti-P-Tyr). (b) Celllysates derived as in panel a were sequentially immunoprecipitated withanti-2B4, anti-NTB-A, anti-NKp46, and anti-IRp60. Equal amounts of eachimmunoprecipitate were analyzed under reducing conditions in 8% SDS-PAGEand probed with either anti-SHP-1 or anti-SHP-2 mAbs. (c) Cell lysatesderived as in panel a were sequentially immunoprecipitated withanti-2B4, anti-NTB-A, and anti-SH2D1A. Samples were analyzed underreducing conditions in 14% SDS-PAGE and probed with anti-SH2D1Aantiserum. In each panel molecular weight markers (kD) are indicated.

FIG. 4. Functional analysis of NTB-A molecules in NK cells derived fromXLP patients. (a) Polyclonal NK cell population derived from XLP patientA was analyzed for cytolytic activity in a redirected killing assayagainst the (FcgR+) P815 target cell line either in the absence or inthe presence of mAbs specific for the indicated molecules. The E/T ratioused was 8:1. All the mAbs used in this experiment were of the IgG1isotype. (b) Polyclonal NK cell populations derived from XLP patient Aor from a healthy donor were analyzed for cytolytic activity against theLCL 721.221 EBV cell line (HLA class I CD48 FcgR+) either in the absenceor in the presence of mAbs specific for the indicated molecules. The E/Tratio used was 6:1. The results are representative of six independentexperiments. The standard deviation of the mean of the triplicates was5%.

FIG. 5. Surface expression of NTB-A in COS-7 cells transfected with theVR1012/KALI construct. COS-7 cells transfected with VR1012/KALIconstruct were stained with MA127 (anti-NTB-A) or PP35 (anti-2B4) mAbsfollowed by PE-conjugated goat anti-mouse second reagent and analyzed byflow cytometry. White profiles indicate cells incubated with the secondreagent only.

FIG. 6. (A) Amino acid sequence of NTB-A molecule (SEQ ID NO:2). Theputative signal peptide is indicated in lower case letters (residues1-21) and the trans-membrane region is boxed. Tyrosine-based motifspresent in the cytoplasmic tail are underlined. (B) Nucleic acid andamino acid sequence of NTB-A molecule (SEQ ID NO:1).

BRIEF DESCRIPTION OF THE SEQUENCES

-   -   SEQ ID NO:1 is a nucleic acid and amino acid sequences of the        NTB-A molecule.    -   SEQ ID NO:2 is an amino acid sequence of the NTB-A molecule.    -   SEQ ID NO:3 is a primer KALI-up sequence.    -   SEQ ID NO:4 is a primer KALI-down sequence.

MATERIALS AND METHODS

Monoclonal Antibodies (m Abs)

MA127 mAb was obtained by immunizing a 5-wk-old BALB/c mouse with the NKclone KK4 (surface phenotype: CD3 CD16 CD56 NCR CD94/NKG2A) as describedpreviously (6, 8, 9). The following mAbs, produced in our lab, were usedin this study: JT3A (IgG2a, anti-CD3), BAB281 and KL247(IgG1 and IgM,respectively, anti-NKp46), Z231 and KS38(IgG1 and IgM, respectively,anti-NKp44), Z25 and F252 (IgG1 and IgM, respectively, anti-NKp30), PP35and S39 (IgG1 and IgG2a, respectively, anti-2B4), KD1 and c127 (IgG2aand IgG1, respectively, anti-CD16), c218 and GPR165 (IgG1 and IgG2a,respectively, anti-CD56), A6-136 (IgM, anti-HLA class I), XA185 (IgG1,anti-CD94), and Z199 and Z270 (IgG2b and IgG1, respectively,anti-NKG2A). D1.12 (IgG2a, anti-HLA-DR) mAb was provided by Dr. R. S.Accolla (Pavia, Italy). HP2.6 (IgG2a, anti-CD4) mAb was provided by Dr.P. Sanchez-Madrid (Madrid, Spain). WT31 (IgG1, anti-TCR-/) was purchasedby Becton Dickinson.

Purification of Polyclonal or Clonal NK Cell Populations

To obtain PBLs, PBMCs were isolated on Ficoll-Hipaque gradients anddepleted of plastic-adherent cells (6). Enriched NK cells were isolatedby incubating PBLs with anti-CD3 (JT3A), anti-CD4 (HP2.6), andanti-HLA-DR (D1.12) mAbs (30 min at 4C) followed by goat anti-mousecoated Dynabeads (Dynal; 30 min at 4C) and immunomagnetic depletion (6).CD3 CD4 DR cells were cultured on irradiated feeder cells in thepresence of 100 U/ml rIL-2 (Proleukin; Chiron Corp.) and 1.5 ng/ml PHA(GIBCO BRL) in order to obtain polyclonal NK cell populations or, afterlimiting dilution (6, 8), NK cell clones.

XLP Patients

The XLP patients analysed in this study are affected by mutations at theSH2D1A locus represented by: G to T nucleotide change at the translationinitiation codon (ATG to ATT), leading to a motioning to isoleucineamino acid change (patient A) and C to T nucleotide change at position163, leading to premature termination at codon 55 (patients B and C). Asdescribed previously (28), these mutations result in a complete absenceof SH2D1A protein.

Cytolitic Activity and Flow Cytofluorimetric Analysis

NK cells were tested for cytolytic activity against the (FcR) P815murine mastocytoma cell line or the lymphoblastoid cell line (LCL)721.221 EBV cell line (HLA class I CD48 FcR) in a 4-h 51 Cr-releaseassay as described previously (6, 8, 9, 28). The concentrations of thevarious mAbs added were 0.5 g/ml for redirected killing or 10 g/ml formasking experiments. The E/T ratios are indicated in the text. For one-or two-color cytofluorimetric analysis (FACSCAN; Becton Dickinson) cellswere stained with the appropriate mAbs followed by PE- orFITC-conjugated isotype-specific goat anti-mouse second reagent(Southern Biotechnology Associates, Inc. [6, 8, 9]).

Biochemical Characterization of NTB-A Molecules

Cells were labeled with Biotin (Pierce Chemical Co.) as describedpreviously (28). 1% NP-40 cells lysates were immunoprecipitated withSepharose-PA (Amersham Pharmacia Biotech)-coupled mAbs. Samples wereanalyzed by discontinuous SDS-PAGE either undigested or digested withN-glycosidase F (Boehringer; reference 28) and transferred to ImmobilonP (Millipore). After staining with Neutravidin (Pierce Chemical Co.),the Renaissance Chemiluminescence Kit (NEN Life Science Products) wasused for detection.

Analysis of the NTB-A Signal Transduction Pathway

NK cells (10⁸) were stimulated or not with 100 M sodium pervanadate(28). 1% NP-40 or 1% digitonin cell lysates were immunoprecipitated withSepharose-PA (Amersham Pharmacia Biotech)-coupled mAbs. Samples wereanalyzed in discontinuous SDS-PAGE, transferred to Immobilon P(Millipore), and probed with: (a) anti-phosphotyrosine mAb (PY20-HRPO;Transduction Laboratories); (b) anti-SHP-2 or anti-SHP-1 mAbs (PTP1D andPTP1C, respectively; Transduction Laboratories) followed by rabbitanti-mouse-HRPO (Dako); (c) anti-SH2D1A rabbit anti-serum (produced byEurogentec S.A; reference 28) followed by donkey anti-rabbit-HRPO(Amersham Pharmacia Biotech). The Renaissance Chemiluminescence Kit (NENLife Science Products) was used for detection. Putative NTB-Aassociation with known signal transducing molecules was analyzed byprobing NTB-A immunoprecipitates, obtained from 1% digitonin celllysates, with anti-FcRI (provided by E. Vivier, Marseille, France),anti-DAP12 (SI-28; reference 10), and anti-LAT (UBI; UpstateBiotechnology) antisera or with anti-CD3mAb (TIA/2; Immunotech).

Library Screening by cDNA Expression in COS-7 and Sib Selection

The library screening was performed as described previously (7). Inbrief, the cDNA library, fractionated in 10 different pools, wastransiently transfected in COS-7 cells using non liposomal FuGene-6reagent (Roche) following the manufacturer's instruction. Selection ofpositive pools was performed by immuno-cytochemical staining using thespecific anti-NTB-A mAb MA127 and sib selection.

DNA Sequencing and Reverse Transcription PCR Analysis

DNA sequencing was performed using d-Rhodamine Terminator CycleSequencing kit and a 377 ABI automatic sequencer (PerkinElmer/AppliedBiosystems). The analysis of the putative protein coded by the KALI cDNAwas performed using the GeneWorks 2.5.1N and see Worldwide Website:genome.cbs.dtu.dk/htbin/ nph-webface. RNA, extracted using RNAzol(Cinna/Biotecx), and oligo (dT)-primed cDNA were prepared frompolyclonal NK cell populations and thymocytes by standard techniques.The set of primers KALI-up (containing the ATG initiation codon): 5′ GCGGAA AGC ATG TTG TGG (SEQ ID NO: 3) and KALI-down (designed in the 3untranslated region): 5′ TCA TTC CCG AAT TCC TCT G (SEQ ID NO: 4) wereused to amplify the KALI-ORF. 30 cycles PCR (30 s, 95° C.; 30 s, 60° C.;and 30 s, 72° C.) was performed using TAQ-GOLD (PerkinElmer/AppliedBiosystems) after preactivation of 12 min at 95° C. The obtainedamplification products were cloned into pcDNA3.1/V5/His-TOPO(topoisomerase) vector using the Eukaryotic-TOPO-TA (topoisomerase)Cloning kit (Invitrogen) and sequenced.

Transient Transfection

COS-7 cells (5.10⁵/plate) were transfected with VR1012/KALI constructusing FuGene-6 reagent (Roche; reference 23). After 48 h, transfectedcells were stained with MA127 (anti-NTB-A) and PP35 (anti-2B4; asnegative control) mAbs followed by Ig-G1 PE-conjugated goat anti-mousesecond reagent and analyzed by flow cytometry using a FACSORT (BectonDickinson).

Chromosomal Localization and Zoo-Blot Analysis

The Somatic Cell Hybrid blot (BIOS Laboratories), containing 20multichromosomal somatic human/hamster cell hybrids plus 3 controlgenomic DNAs (human, hamster, and mouse) was used to assign the KALIgene to a specific chromosome (10). The open reading frame of KALI genewas used as probe to perform high stringency hybridization (37).Analysis of cross-specific conservation of KALI gene was performed usingZoo-Blot from CLONTECH Laboratories, Inc. This Southern blot containedgenomic DNA from human, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse,dog, cow, rabbit, chicken, and Saccharomyces cerevisiae yeast. Washeswere carried out under low stringency condition (7).

RESULTS

Identification and Cellular Distribution of NTB-A Molecule

Mice were immunized with the human NK cell clone KK4 (surface phenotype:CD3 CD16 CD56 NCR CD94/NKG2A). After cell fusion, a mAb, termed MA127(IgG1), was selected on the basis of its ability to induce cytotoxicityin polyclonal or clonal NK cells (including the immunizing NK cell cloneKK4) in a redirected killing assay against the FcR P815 murine targetcells (see below). The cell surface distribution of the MA127-reactivemolecules was analyzed by indirect immunofluorescence andcytofluorimetric analysis in peripheral blood mononuclear cells (PBMCs)from normal donors. As shown in Table 1, MA127 mAb brightly stained NK,T, and B lymphocytes. On the other hand, no reactivity was detected withmonocytes, granulocytes, and a panel of nonlymphoid cell lines.Polyclonal populations of either NK cells or TCR-/cells or thymocyteswere surface-labeled with biotin and cell lysates wereimmunoprecipitated with MA127 mAb. In all instances, this mAbimmunoprecipitated a surface molecule of 60 kD both under reducing(FIG. 1) and nonreducing conditions (not shown). The protein back-boneremaining after treatment with N-glycosidase F, displayed a molecularmass of 37 kD. These data suggested that MA127 mAb may recognize a noveltriggering molecule expressed not only by NK cells but also by T and Blymphocytes; this molecule was thereafter termed NK-T-B-antigen (NTB-A).

Clonal Heterogeneity in Response to Anti-NTB-A mAb-MediatedCross-Linking

It has been shown that heterogeneity exists among NK cell clones intheir ability to kill a given HLA class I-negative target cell and thatthis reflects the differential expression of NCRs (12). Thus, NK cellclones displaying strong cytolytic activity express high levels of NCRs(NCR bright) whereas those with low cytolytic activity express lowamounts of NCRs (NCR dull). Importantly, the NCR surface density wasalso found to correlate with the function of 2B4. Thus, although 2B4 isexpressed at similar surface densities in both types of NK cell clones,it induces cytotoxicity only by the NCR bright ones (23). A panel of NKcell clones displaying either NCR bright or NCR dull phenotype wasanalyzed in a redirected killing assay against P815 target cells in thepresence of MA127 mAb. As shown in FIG. 2 a, whereas NKp46 bright cloneswere responsive both to anti-NCR and anti-2B4 or anti-NTB-A mAbs, NKp46dull clones responded poorly to all of these mAbs (although they didrespond efficiently to anti-CD16 mAb). Remarkably, all the NK cellclones analyzed expressed similar surface densities of NTB-A (notshown). To further explore the relationship between responsiveness toanti-NTB-A mAb and surface density of NKp46, we analyzed the effect ofsurface modulation of NKp46 molecules. To this end, NKp46 bright cloneswere incubated overnight in the presence of immobilized anti-NKp46 mAb(KL247, IgM). This resulted in a virtually complete disappearance ofNKp46 molecules from the cell surface (23; data not shown). Thistreatment did not affect the surface expression of NTB-A or of other NKcell surface molecules. NKp46-modulated clones were used as effectors ina redirected killing assay against P815 target cells in order to analyzetheir responsiveness to mAbs directed to different surface molecules. Inagreement with previous data indicating that NKp46 functions as themajor human receptor in the recognition and lysis of murine targets (7,12, 38), NKp46-modulated NK cells displayed a sharply reduced ability tospontaneously lyse P815 cells (in the absence of mAbs; FIG. 2 b;reference 23). Importantly, the analysis of the effect of different mAbsrevealed that the unresponsiveness was limited not only to anti-NKp46 oranti-2B4 mAbs (23) but also to anti-NTB-A mAb. On the contrary,responses to anti-NKp44, anti-NKp30, or anti-CD16 mAb were not affected(23). These data are reminiscent of previous data using anti-2B4 mAbs(23) and suggest that also NTB-A may function as a coreceptor in themechanism of NK cell activation. Note, however, that both the cellsurface distribution and the molecular mass of NTB-A are clearlydistinct from those of 2B4 (see Table 1 and FIG. 1).

Involvement of NTB-A in NK-Mediated Killing of EBV Target Cells

2B4 has been shown to cooperate with NKp46 in the process of recognitionand killing of EBV-infected cells such as the (HLA class I CD48 FcR) LCL721.221 (28). On the contrary, the contribution of other triggeringreceptors expressed by NK cells, including NKp30, NKp44, and NKG2D, inNK-mediated lysis of LCL 721.221 is marginal or even absent (data notshown). We analyzed whether also NTB-A is involved in lysis of thesetarget cells. Thus, NK cell clones, derived from normal donors, wereassessed for cytolytic activity against the LCL 721.221 (FIG. 2 c) orDaudi Burkitt lymphoma (not shown) cell lines, either in the absence orin the presence of mAbs directed to NTB-A, 2B4, or NKp46. mAb-mediatedblocking of either NTB-A or 2B4 did not significantly affect theNK-mediated killing of these targets. However, the combined use of mAbsdirected to NTB-A and 2B4 resulted in a partial inhibition of lysis.Importantly, the inhibitory effect obtained by mAb-mediated masking ofNKp46 was significantly incremented in the presence of either anti-NTB-Aor anti-2B4 mAbs and maximal inhibition was obtained when the threemolecules were simultaneously blocked by the respective mAbs.Isotype-matched anti-CD56 mAb used either alone or in combination had noinhibitory effect (not shown). These data suggest that, in normal cells,NTB-A, together with 2B4, cooperates with NKp46 in the induction ofNK-mediated cytotoxicity against EBV target cells. Moreover, theysuggest that NTB-A, similar to 2B4, recognizes cell surface ligand(s)expressed on EBV targets. However, it should be stressed that dataobtained using normal NK cells may be of difficult interpretationbecause different receptors (such as NKp46) and coreceptors (such asNTB-A and 2B4) are involved in the cytolytic activity against EBVtargets. On the other hand, that NTB-A, similar to 2B4 (28), is indeedcapable of recognizing EBV target cells was confirmed by experiments inwhich the NK92 leukemic NK cell line was used as source of effectorcells. This cell line is NTB-A 2B4 but expresses very low amounts ofNKp46 (not shown). Accordingly, mAb-mediated cross-linking of NKp46fails to induce NK92-mediated cytotoxicity against P815 in a redirectedkilling assay (FIG. 2 d). Importantly however, in this assay thecytotoxicity of NK92 cells could be strongly enhanced by mAb-mediatedcross-linking of either NTB-A or 2B4. These data suggest that in thecase of NK92 cells (different from normal NK cells) the NTB-A and2B4-mediated activation was mostly NKp46 independent. Thus, NK92 cellline offered a useful tool to clarify the involvement of NTB-A in therecognition and killing of EBV target cells. Indeed, as shown in FIG. 2d, NK92 cells were strongly cytolytic against LCL 721.221 target cells.mAb-mediated masking of either NTB-A or 2B4 resulted in a consistentinhibition of cytotoxicity whereas the combined use of both mAbsresulted in the virtual abrogation of NK92-mediated killing of LCL721.221. Altogether, these data support the notion that NTB-A isinvolved in the recognition of EBV targets.

Analysis of the NTB-A-Mediated Signal Transduction Pathway

NTB-A molecules were immunoprecipitated from polyclonal NK cellpopulations treated or not with sodium pervanadate. Tyrosinephosphorylation of NTB-A molecules was consistently detectable inimmunoprecipitates obtained from sodium pervanadate-treated but not fromuntreated cells (FIG. 3 a). On the contrary, NTB-A did not associatewith signal transducing polypeptides, including CD3, FcRI, and DAP12(not shown). These data suggested that, different from NCRs but similarto 2B4, NTB-A molecules may display tyrosine-based motifs in theirintracytoplasmic portion. These motifs were likely to mediate theassociation with intracytoplasmic molecules involved in the transductionof activating signals. To analyze this possibility, NTB-Aimmunoprecipitates, obtained as above, were probed with anti-SH2D1Aantiserum or with mAbs specific for SHP-1 or SHP-2. As a control,identical cell lysates were immunoprecipitated with mAbs specific for2B4, or for the inhibitory receptor protein 60 (IRp60) or NKp46. 2B4 hasbeen shown to associate with SHP-1 and, upon sodium pervanadatetreatment or mAb-mediated cross-linking, with SH2D1A (27-29). On theother hand, IRp60 is an immune tyrosine-based inhibitory motif(ITIM)-bearing receptor that associates with both SHP-1 and SHP-2 (39),while NKp46, lacking tyrosine-based motifs in the cytoplasmic tail,transduces the activating signals via the association with CD3 and FcRIITAM-containing transmembrane adaptor molecules (4). This analysisrevealed the presence of SHP-1 in NTB-A immunoprecipitates obtained fromboth untreated and sodium pervanadate-treated cells. In contrast, theassociation of NTB-A with SHP-2 could be detected only in treated cells(FIG. 3 b). Notably, treatment with sodium pervanadate also led to theassociation of NTB-A with SH2D1A molecules (FIG. 3 c). Altogether, thesedata strengthened the idea that NTB-A molecules, although exhibiting adistinct surface distribution in PBMCs, display functional and molecularcharacteristics similar to 2B4.

NTB-A Mediates Inhibitory Rather than Activating Signals in XLP-NK Cells

In view of the similarities with 2B4, we analyzed the function of NTB-Ain polyclonal and clonal NK cells from different patients affected byXLP (see Materials and Methods). XLP-NK cells were analyzed in aredirected killing assay against P815 murine targets either in theabsence or in the presence of mAbs specific for various surfacemolecules including NTB-A and 2B4. As shown in FIG. 4 a, and inagreement with previous data (28), in NK cells from the representativeXLP patient A, 2B4 exhibited inhibitory rather than activating functionwhereas CD16 (not shown) and NCRs displayed normal triggeringcapability. Importantly, cross-linking of NTB-A by specific mAb alsoresulted in a marked inhibition of spontaneous cytotoxicity. An evengreater inhibitory effect was observed in the presence of both NTB-A-and 2B4-specific mAbs (FIG. 4 a). Moreover, cross-linking of NTB-A wasable to inhibit the cytolytic activity induced by anti-CD16 (not shown)or anti-NCR mAbs. Consistent with previous results (28), a similarinhibitory activity was observed in the presence of anti-2B4 mAb.Although not shown, the absence of SH2D1A in XLP-NK cells did not affectthe level of expression of NTB-A at the cell surface. Based on theobservation that NTB-A, similar to 2B4, may recognize cell surfaceligand(s) expressed on EBV target cells (see above) we asked whether inXLP-NK cells this interaction could induce the generation of inhibitorysignals via NTB-A. XLP-NK cells were further analyzed for their abilityto lyse the (HLA class I FcR) LCL 721.221 EBV cell line expressing largeamounts of CD48 (i.e., the natural ligand of 2B4). In agreement withprevious report (28), these target cells were efficiently lysed bynormal NK cells while they were resistant to lysis by XLP-NK cells (FIG.4 b). mAb-mediated masking of either 2B4 (28) or NTB-A moleculesresulted in partial restoration of lysis of LCL 721.221 cells; thiseffect was further incremented by the simultaneous masking of bothmolecules (FIG. 4 b). It is of note that, unlike 2B4, NTB-A molecule isexpressed by both effectors (NK cells) and targets (B cells). However,restoration of lysis could be detected only upon pretreatment ofeffector but not of target cells with anti-NTB-A mAb (not shown).Similar results were obtained in XLP-NK cells derived from patient B(28) and patient C carrying the same mutation (not shown).

Thus, the lack of SH2D1A in XLP patients resulted in a profounddysfunction not only of 2B4 but also of the newly identified NTB-Amolecule. Accordingly, both molecules appear to contribute to theinability of XLP-NK cells to kill EBV target cells.

Molecular Cloning and Characterization of the cDNA Encoding the NTB-ASurface Molecule

The cDNA encoding NTB-A molecule was isolated from an NK cell-derivedcDNA expression library using MA127 mAb (7). This cDNA (referred asKALI) was characterized by a length of 2,744 bp. Transfection of theVR1012/KALI construct in COS-7 cells allowed the surface expression ofmolecules that were brightly stained by MA127 mAb (FIG. 5). Moreover,MA127 mAb specifically immunoprecipitated from cell transfectants aprotein that, after treatment with N-glycosidase F, displayed a proteinbackbone identical to that of NTB-A molecules derived from polyclonal NKcells (not shown). The predicted amino acid sequence is consistent witha type I transmembrane protein of 331 amino acids belonging to the Ig-SF(FIG. 6). The NTB-A molecule is characterized by a 21 amino acid leaderpeptide preceding an extracellular region of 204 residues. Proteinsequence analysis suggests that the extracellular portion is composed bya N-terminal V-type domain (lacking the classical disulfide bond)followed by a C2-type domain (characterized by two possible intradomaindisulfide bonds). The extracellular portion contains seven potentialN-linked glycosylation sites, but no putative O-glycosylation sites. A23 amino acid long transmembrane region, lacking charged amino acidresidues, precedes a relatively long (83 amino acids) intracytoplasmicportion that contains three tyrosine residues. Two tyrosine residues arepart of the TxYxxV/I motif that has recently been described in the 2B4cytoplasmic tail (25-28). Notably, these motifs are believed torepresent consensus sequence for the association with SH2D1A. The thirdtyrosine residue is included in a classical ITIM (I/V/L/SxYxxL/V;reference 3) that is absent in the 2B4 cytoplasmic tail. Reversetranscription (RT)-PCR performed on RNA derived from different NK cellclones using KALI ORF-specific primers revealed the existence of asecond cDNA coding for a putative allelic isoform of NTB-A. This cDNA ischaracterized by an extra codon (CAG) resulting in the insertion of anAla residue at position 266 of the mature protein. Comparison of theNTB-A amino acid sequence in GenBank/EMBL/DDBJ database with otherpreviously identified proteins revealed homology with CD84 (40) (25% ofidentity). Chromosomal localization by Southern blot analysis revealedsegregation of NTB-A encoding gene on human chromosome 1 (not shown).Remarkably, the same chromosomal localization has been established alsofor all the genes coding for the various members of the CD2 subfamily(24). This finding, together with the particular core structure of theIg-like domains (characterized by an N-terminal non-disulfide-bondedIg-V domain and by a membrane-proximal Ig-C2 domain containing twopossible intradomain disulfide bonds) suggests that NTB-A represents anovel member of the CD2 subfamily. Finally, hybridization of zooblotwith KALI ORF probe suggested a cross-species conservation between humanand monkey (not shown).

Discussion

In this study we have identified, molecularly characterized, and clonedNTB-A, a novel triggering surface molecule belonging to the CD2subfamily, that is expressed on resting and activated lymphoidpopulations including NK, T, and B lymphocytes. Similar to 2B4 (23),triggering of normal NK cells via NTB-A requires the simultaneousengagement of NKp46. The role of NTB-A as a co-receptor is furtherdocumented by mAb-mediated masking experiments. In these experiments,the lysis of EBV-infected B cell lines mediated by normal NK cells wasinhibited by the simultaneous masking of NTB-A, NKp46, and 2B4.Biochemical analysis revealed that NTB-A, similar to 2B4 (27, 28),associates with SH2D1A and SHP. SH2D1A is a small intracytoplasmicadaptor molecule the expression of which appears to be highly regulated.Indeed, very low levels of SH2D1A can be detected in resting cells whilethey dramatically increase upon cell activation (41). It is unlikelythat SH2D1A may play a direct role in co-stimulation. On the other hand,it is likely that SH2D1A simply participates to the transduction ofNTB-A-mediated activating signals by competing with the intracytoplasmicphosphatases for binding to this activating coreceptor. Along this line,previous reports showed that SH2D1A is crucial for the transduction ofactivating signals via 2B4 (27, 28) and CD150 (42). Moreover, 2B4tyrosine phosphorylation and association with SH2D1A was detected notonly upon sodium pervanadate treatment but also upon 2B4 mAb-mediatedcross-linking (29). The finding that NTB-A associates upon tyrosinephosphorylation with SH2D1A led us to investigate the function of NTB-Ain individuals affected by XLP (33, 34). XLP is characterized bycritical mutations in the SH2D1A encoding gene (30, 35, 36). XLPpatients suffer from a severe immunodeficiency resulting in theinability to control EBV infections. Previous studies suggested thatSH2D1A-associated molecules may play an important role in the failure ofcytolytic cells to kill EBV-infected target cells. In this context, 2B4molecule, which functions as a triggering coreceptor in normal NK cells(23), has been shown to display a profound alteration of the signalingpathway in the case of XLP-NK cells. Thus, due to the absence of SH2D1Aassociation, 2B4 displays an opposite function, i.e., mediatesinhibitory rather than activating signals. 2B4 engagement either byspecific mAb or by its natural ligand (CD48) expressed at high densityin EBV-infected cells, resulted in down regulation of XLP-NKcell-mediated cytotoxicity. This was true both for the spontaneouscytolytic activity and for the NK cell triggering induced via differentactivating receptors (i.e., NKp46; reference 28, and this report).Analysis of the amplified cDNA obtained by RT-PCR in polyclonal XLP-NKcells revealed, in all samples analyzed, that the NTB-A sequence isidentical to that obtained from healthy donors (not shown). Moreover,cytofluorimetric analysis of XLP-NK cells showed that the absence ofSH2D1A molecule does not affect the surface expression of NTB-A.Importantly, in XLP-NK cells NTB-A appears to play a role similar to 2B4(28). Indeed, in the absence of SH2D1A, NTB-A does not transducetriggering but rather inhibitory signals. This can easily be appreciatedin redirected killing assays in which mAb-mediated cross-linking ofNTB-A results in inhibition of both the spontaneous and the NCR-mediatedcytotoxicity of XLP-NK cells. More importantly, similar inhibitorysignals were generated when XLP-NK cells interacted with EBV targetcells. In addition to the 2B4/CD48 interaction (28), another inhibitorysignal was generated by the interaction between NTB-A and still unknownligand(s) expressed on EBV cells. Thus, mAb-mediated blocking of NTB-Apartially restored the XLP-NK cell-mediated lysis of EBV targets whileit had no effect on the lysis of different tumor targets includingmelanomas, lung carcinomas, T cell lymphomas, and cervical carcinomas(not shown). This suggests that, similar to CD48, the expression ofNTB-A ligand(s) may be restricted to certain cell types. Alternatively,only some cells may express sufficient surface densities of theligand(s) to allow signalling upon binding with NTB-A. It is possiblethat NTB-A similar to CD150 (42) may display homophilic interactions orinteract with other members of the CD2 subfamily (as in the case ofCD2/CD48, CD2/CD58, and 2B4/CD48 interactions) (21, 22, 43). Preliminarydata would suggest that the ligand for NTB-A is not represented by CD48(i.e., the 2B4 ligand). Thus, upon simultaneous masking of both NTB-Aand 2B4, an additive effect occurs in the restoration of XLP-NKcell-mediated lysis of EBV target cells (see above). Moreover, whereasonly a partial restoration of NK-mediated cytotoxicity occurred uponmAb-mediated masking of CD48 on EBV target cells, a strong increment oflysis could be detected by the simultaneous masking of NTB-A on XLP-NKcells (data not shown). In this study the analysis of the cytolyticactivity of XLP-NK cells against EBV targets has been mainly evaluatedagainst HLA class I EBV LCL in order to avoid interference due to killerinhibitory receptor (KIR)/HLA class I interactions. We previouslyshowed, in XLP patients, that the recovery of NK-mediated cytotoxicityagainst autologous HLA class I EBV LCL cells did not occur uponmAb-mediated disruption of HLA/KIR interactions. Unlike normal donors,in these patients, only the simultaneous mAb-mediated masking of HLAclass I and 2B4 led to reconstitution of cytotoxicity (28). Although notshown, similar results could be obtained by the simultaneous masking ofHLA class I and NTB-A. Thus, unlike in normal individuals, in XLPpatients, clearance of HLA-deficient EBV-infected cells would beimpaired because of the occurrence of inhibitory rather than activatinginteractions between 2B4, NTB-A, and their ligands. In this context,different studies reported that down regulation of HLA class I moleculesoccurs during EBV infection (44-46). However, this event may occur “invivo” only at given stages of EBV infection and/or it may affect one orfew HLA class I alleles. Indeed, although most EBV-infected cells mayresult HLA class I when analyzed with mAbs directed to frameworkdeterminants of HLA class I, this analysis is clearly inadequate toreveal a single allelic loss. It should be stressed that down regulationof a single allele renders target cells susceptible to NK cellsexpressing KIR specific for the missing allele (1). In other reports noinhibitory function of 2B4 has been observed in XLP-NK cells. Thesestudies suggested a “lack of function” of 2B4 (31, 32). Further studiesshould clarify these divergent results and in particular whether theyreflect a heterogeneity of the patients' phenotype or other yet unknownmechanisms. We are presently investigating three additional XLP patientscarrying mutations at the SH2D1A locus different from those detected inpatients A, B, and C. Preliminary data suggest that also in thesepatients both 2B4 and NTB-A display inhibitory functions (data notshown). Thus, in six different XLP patients we could obtain consistentresults. These findings may be of particular relevance, as disruption ofthe interaction between 2B4 or NTB-A with their ligands may lead torestoration of cytolytic function. This may have important implicationsfor therapy of otherwise fatal acute EBV infections in XLP patients. Assuggested by the analysis of T cell mediated cytolytic activity in aredirected killing assay, NTB-A fails to trigger cytotoxicity in thesecells (data not shown). Also, this finding is reminiscent of previousdata on 2B4 molecules (28). Notably, T cells do not express the NKp46receptor specific for ligand(s) present on murine cells such as the P815target cell used in redirected killing assays. Thus, CTLs may beunresponsive to mAbs specific for 2B4 and NTB-A in this experimentalsetting, simply because both molecules act as coreceptors and requirefor their function a costimulus provided by a true receptor (e.g.,NKp46). On the other hand, it is conceivable that, in T cells, othertriggering receptors may be physiologically involved in providing thesignal required for 2B4- and NTB-A signalling. Along this line, furtherstudies should clarify whether EBV-specific CTLs actually use 2B4 andNTB-A as co-receptors and whether these molecules may inhibit Tcell-mediated responses against EBV in XLP patients. Regarding thefunction of NTB-A in B lymphocytes, studies are in progress in order toclarify this issue. It is of note that so far no SH2D1A could bedetected in normal B cells as well as in most B cell lines (41). Thus,it is likely that, in order to transduce signals in B cells, NTB-A mayrequire association with a distinct intracytoplasmic adaptor molecule.In this context, a possible candidate is represented by the recentlydescribed EAT-2 molecule that is expressed in B cells and displays highidentity with SH2D1A (41). Molecular cloning revealed that NTB-Arepresents a novel member of the CD2 subfamily (24). Although thesemolecules display a relatively limited amino acid identity, they areclustered on human chromosome 1 and display a remarkably similar corestructure of the Ig-like domains. NTB-A does not contain classical ITAMconsensus sequence in the cytoplasmic tail. Different from variousactivating surface receptors but similar to 2B4 (8-10, 16), NTB-A doesnot associate with DAP12, CD3, and FcRI signal transducing polypeptides.Moreover, unlike 2B4 (29), NTB-A does not associate with the LAT (notshown). In this context, sequence analysis revealed that NTB-A lacksboth the charged amino acid residues in the transmembrane region and theC×C/motif (i.e., a C×C sequence surrounded by positive-charged residues)in the transmembrane/cytoplasmic portion. These motifs have beensuggested to play a crucial role in the different receptor/adaptorinteractions (4, 47). An additional feature common to 2B4 (28) is theability of NTB-A to bind SHP-1 and, upon tyrosine phosphorylation, alsoSH2D1A. Consistent with the latter association, the cytoplasmic tail ofNTB-A contains two TxYxxV/I motifs that are thought to representconsensus sequences for the association with SH2D1A (4). Moreover, theamount of SHP-1 associated to NTB-A appears to be reduced upon sodiumpervanadate treatment and SH2D1A binding (see FIG. 3). These data aresimilar to those obtained on 2B4 (28) and suggest that SH2D1A maycompete for binding to SHP-1 also in the case of NTB-A. At variance with2B4, NTB-A is characterized by a classical ITIM motif in the cytoplasmicportion and can associate with SHP-2 upon tyrosine phosphorylation.SHP-2 has been found to associate with both inhibitory and activatingreceptors (48), suggesting that the final functional outcome of therecruitment of SHP-2 may depend on the functional characteristics of thedifferent SHP-2-specific substrates. Thus, so far, the actual role ofthis phosphatase in the NTB-A-mediated signalling remains to bedetermined. TABLE 1 Surface Expression of MA127-reactive molecules MA127Anti-2B4 Cells Histotype mAb mAb Resting NK cells + + Activated NKcells + + Resting T cells + + (subset) PHA Blasts + + (subset) Resting Bcells + − Thymocytes + + Monocytes − + Granulocytes − − YT NK cellline + + NKL NK cell line + + NK3.2 NK cell line + + NK92 NK cellline + + JA3 T leukemia + + H9 T leukemia + − HSB2 T leukaemia + + RajiBurkitt lymphona + − DAUDI Burkitt lymphona + − LCL 721.221 EBV-Cellline + − U937 histiocytic lymphona − + HL60 promyelocityc leukemia − +TF1 promyelocityc leukemia − + MM6 promyelocityc leukemia − + Eo/A3Eosinophilic leukemia − + MEL15392 melanoma − − MEL501 melanoma − − F0-1melanoma − − 1074 mel melanoma − − A549 lung carcinoma − − SMMC hepatoma− − HELA cervical carcinoma − − IGROV-1 cervical carcinoma − − YAC-1murine thymoma − − BW1502 murine thymoma − − P815 murine mastocytoma − −COS-7 monkey kidney fibroblast − −

Normal cells and tumor cell lines of different histotype were analysedby immunofluorescence and FACS analysis for reactivity with MA127 andPP35 (anti-2B4) mAbs followed by PE-conjugated goat anti-mouse IgG1.Cells are of human unless otherwise specified.

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1. A composition of matter comprising: a) an isolated nucleic acidmolecule, wherein said nucleic acid molecule is selected from: i) anucleic acid encoding a polypeptide comprising SEQ ID NO:2; ii) anucleic acid which hybridizes to the nucleic acid of a) or to a portionthereof, said portion comprising at least 30 contiguous nucleotides of anucleic acid i); iii) a complementary strand of a nucleic acid moleculeof i) or ii); iv) a nucleic acid molecule encoding a polypeptidecomprising at least 5 contiguous amino acid residues of SEQ ID NO:2; v)a nucleic acid molecule that encodes a human NTB-A protein; or vi) afragment of a nucleic acid molecule of i), ii), iii), iv) or v), saidfragment comprising at least 9 contiguous nucleotides; b) a nucleic acidprobe, wherein said probe is complementary and specifically hybridizesto a nucleic acid molecule selected from: i) a nucleic acid encoding apolypeptide comprising SEQ ID NO:2; ii) a nucleic acid which hybridizesto the nucleic acid of a) or to a portion thereof, said portioncomprising at least 30 contiguous nucleotides of a nucleic acid i); iii)a complementary strand of a nucleic acid molecule of i) or ii); iv) anucleic acid molecule encoding a polypeptide comprising at least 5contiguous amino acid residues of SEQ ID NO:2; v) a nucleic acidmolecule that encodes a human NTB-A protein; or vi) a fragment of anucleic acid molecule of i), ii), iii), iv) or v), said fragmentcomprising at least 9 contiguous nucleotides; c) a pair of nucleic acidprimers, wherein at least one primer of said pair is complementary andspecifically hybridizes to a nucleic acid molecule selected from: i) anucleic acid encoding a polypeptide comprising SEQ ID NO:2; ii) anucleic acid which hybridizes to the nucleic acid of a) or to a portionthereof, said portion comprising at least 30 contiguous nucleotides of anucleic acid i); iii) a complementary strand of a nucleic acid moleculeof i) or ii); iv) a nucleic acid molecule encoding a polypeptidecomprising at least 5 contiguous amino acid residues of SEQ ID NO:2; v)a nucleic acid molecule that encodes a human NTB-A protein; or vi) afragment of a nucleic acid molecule of i), ii), iii), iv) or v), saidfragment comprising at least 9 contiguous nucleotides; d) a vectorcomprising a nucleic acid molecule selected from: i) a nucleic acidencoding a polypeptide comprising SEQ ID NO:2; ii) a nucleic acid whichhybridizes to the nucleic acid of a) or to a portion thereof, saidportion comprising at least 30 contiguous nucleotides of a nucleic acidi); iii) a complementary strand of a nucleic acid molecule of i) or ii);iv) a nucleic acid molecule encoding a polypeptide comprising at least 5contiguous amino acid residues of SEQ ID NO:2; v) a nucleic acidmolecule that encodes a human NTB-A protein; or vi) a fragment of anucleic acid molecule of i), ii), iii), iv) or v), said fragmentcomprising at least 9 contiguous nucleotides; e) a host cell comprisinga vector, said vector comprising a nucleic acid molecule selected from:i) a nucleic acid encoding a polypeptide comprising SEQ ID NO:2; ii) anucleic acid which hybridizes to the nucleic acid of a) or to a portionthereof, said portion comprising at least 30 contiguous nucleotides of anucleic acid i); iii) a complementary strand of a nucleic acid moleculeof i) or ii); iv) a nucleic acid molecule encoding a polypeptidecomprising at least 5 contiguous amino acid residues of SEQ ID NO:2; v)a nucleic acid molecule that encodes a human NTB-A protein; or vi) afragment of a nucleic acid molecule of i), ii), iii), iv) or v), saidfragment comprising at least 9 contiguous nucleotides; f) an isolatedpolypeptide comprising: i) an amino acid sequence encoded by a nucleicacid molecule according to a); ii) SEQ ID NO:2; or iii) at least 5contiguous amino acid residues of SEQ ID NO:2; g) an isolated antibodythat specifically binds to a polypeptide comprising: i) an amino acidsequence encoded by a nucleic acid molecule according to a); ii) SEQ IDNO:2; or iii) at least 5 contiguous amino acid residues of SEQ ID NO:2;or h) a pharmaceutical composition comprising a compound that regulatesthe activity of NTB-A and a pharmaceutically acceptable vehicle orcarrier.
 2. The composition of matter according to claim 1, wherein saidcomposition of matter is a nucleic acid molecule that comprises asequence encoding a polypeptide comprising SEQ ID NO:2 or a polypeptidecomprising at least 5 contiguous amino acid residues of SEQ ID NO:2. 3.The composition of matter according to claim 1, wherein said compositionof matter is a pharmaceutical composition comprising a compound thatregulates the activity of NTB-A and a pharmaceutically acceptablevehicle or carrier that further comprises a compound that regulates a2B4 receptor.
 4. The composition of matter according to claim 3, whereinthe compound that regulates the activity of NTB-A comprises an antibodythat inhibits or reduces NTB-A activity and the compound that regulatesa 2B4 receptor is an antibody that inhibits or reduces 2B4 activity. 5.A method of using a composition of matter according to claim 1 for: a)the preparation of cells expressing a polypeptide; b) preparing anantibody; c) selecting, screening or characterizing a compound; d)regulating the immune function of a patient; or e) detecting adysfunction in a subject or determining the risk of a subject developinga dysfunction.
 6. The method according to claim 5, wherein said methodis a method of preparing cells expressing a polypeptide, said methodcomprising introducing a nucleic acid molecule or vector into cells invitro and selecting the cells which express the polypeptide or progenyof said cells, wherein said nucleic acid molecule or vector is: a) anucleic acid encoding a polypeptide comprising SEQ ID NO:2; b) a nucleicacid which hybridizes to the nucleic acid of a) or to a portion thereof,said portion comprising at least 30 contiguous nucleotides of a nucleicacid of a); c) a complementary strand of a nucleic acid molecule of a)or b); d) a nucleic acid molecule encoding a polypeptide comprising atleast 5 contiguous amino acid residues of SEQ ID NO:2; e) a nucleic acidmolecule that encodes a human NTB-A protein; f) a fragment of a nucleicacid molecule of a), b), c), d) or e), said fragment comprising at least9 contiguous nucleotides; or g) a vector comprising a nucleic acidmolecule of a), b), c), d), e) or f).
 7. The method according to claim5, wherein said method is a method of preparing an antibody, said methodcomprising injecting to a non-human mammal a polypeptide comprising SEQID NO:2 or a polypeptide comprising at least 5 contiguous amino acidresidues of SEQ ID NO:2 and collecting the antibody, serum orantibody-producing cells in said mammal.
 8. The method according toclaim 5, wherein said method is a method of selecting, screening orcharacterizing a compound, said method comprising contacting a testcompound with a polypeptide comprising SEQ ID NO:2 or a polypeptidecomprising at least 5 contiguous amino acid residues of SEQ ID NO:2 anddetermining the ability of said test compound to bind to saidpolypeptide.
 9. The method according to claim 5, wherein said method isa method of selecting, screening or characterizing a compound, saidmethod comprising contacting a test compound with a host cell anddetermining the ability of said test compound to bind to saidpolypeptide, wherein said host cell comprises a vector, said vectorcomprising a nucleic acid molecule selected from: i) a nucleic acidencoding a polypeptide comprising SEQ ID NO:2; ii) a nucleic acid whichhybridizes to the nucleic acid of a) or to a portion thereof, saidportion comprising at least 30 contiguous nucleotides of a nucleic acidi); iii) a complementary strand of a nucleic acid molecule of i) or ii);iv) a nucleic acid molecule encoding a polypeptide comprising at least 5contiguous amino acid residues of SEQ ID NO:2; v) a nucleic acidmolecule that encodes a human NTB-A protein; or vi) a fragment of anucleic acid molecule of i), ii), iii), iv) or v), said fragmentcomprising at least 9 contiguous nucleotides.
 10. The method accordingto claim 5, wherein said method is a method of selecting, screening orcharacterizing a compound, said method comprising contacting a testcompound with a NK cell in the presence of an antibody specific forNTB-A and determining the activity of said test compound by measuringthe cytolytic activity of said NK cells.
 11. The method according toclaim 5, wherein said method is a method of selecting, screening orcharacterizing a compound, said method comprising contacting a testcompound with a NTB-A polypeptide in the presence of a binding partnerthereof and assessing the capacity of said test compound to modulate theinteraction between said NTB-A polypeptide and said binding partner. 12.The method according to claim 5, wherein said method is a method ofselecting, screening or characterizing a compound, said methodcomprising (i) determining the ability of a test compound to bind to aNTB-A polypeptide and (ii) determining the ability of a test compoundselected in (i) to regulate NK cell-mediated target cell lysis.
 13. Themethod according to claim 5, wherein said method is a method ofregulating the immune function in a subject comprising theadministration of a compound that regulates the activity of a NTB-Apolypeptide to a subject.
 14. The method according to claim 13, whereinthe compound regulates the activity of NK cells in a subject.
 15. Themethod according to claim 5, wherein said method is a method ofdetecting a dysfunction in a subject or the risk of a subject developinga dysfunction comprising determining, in a sample derived from saidsubject, the presence of a mutation or alteration in NTD-A gene or RNA,or determining the presence or amount of a NTB-A polypeptide.